Regulation of the Na, K-ATPase beta1 subunit during TGF-beta2 -mediated epithelial to mesenchymal transition in human retinal pigmented epithelial cells
University of Delaware
Primary rhegmatogenous retinal detachment (RRD) leading to proliferative vitreo retinopathy (PVR) is associated with extracellular matrix membrane (ECM) formation on the neural retina, disruption of the multilayered retinal architecture leading to secondary retinal detachment, distorted vision and blindness. During RRD and PVR, retinal pigmented epithelial cells (RPE) underlying the neural retina lose their cell-cell adhesion with the neural retina and undergo epithelial-tomesenchymal transition (EMT) leading to tissue fibrosis. Cytoskeletal reorganization, expression of mesenchymal proteins and reduced cell-cell adhesion during EMT is mediated by exposure to vitreous cytokines and growth factors such as TGF-ß2 . Studies in our lab have shown that the Na, K-ATPase a and ß subunits play an important role in maintaining a normal polarized epithelial phenotype. Reduced Na, KATPase pump function and Na, K-ATPase ß1 subunit levels are associated with EMT in several epithelial cell types, as well as in cancer. The Na, K-ATPase is apically expressed in RPE in contrast to other epithelia where it is expressed basolaterally. The role played by the Na, K-ATPase subunits in maintaining RPE cell polarity and EMT is currently unknown. This study shows that TGF-ß2 induced a reduction in the membrane and total Na, K-ATPase ß1 protein levels in human retinal pigmented epithelial (ARPE-19) cells. Knockdown of Na, K-ATPase ß1 resulted in mesenchymal morphogenesis, cytoskeletal reorganization and an increase in the EMT marker fibronectin, suggesting that, reduction of Na, K-ATPase ß1 subunit is important for TGF-ß2 -mediated fibrotic EMT in ARPE-19 cells. In addition to protein expression, TGF-ß2 reduced the mRNA levels of Na, K-ATPase ß1 subunit as well. Na, K-ATPase ß1 promoter analysis revealed a putative hypoxia response element (HRE) and a Smad binding domain (SBD). This study shows that the transcription factor HIF-1a was capable of binding to the Na, K-ATPase ß1 promoter and inhibiting HIF-1a DNA binding, blocked the decrease in Na, K-ATPase ß1 expression. However, while HIF-1a might play a potential role in Na, K-ATPase ß1 regulation, factors specific to TGF-ß2 signaling are also required to down regulate Na, K-ATPase ß1 in ARPE-19 cells. These findings demonstrate that the Na, K-ATPase ß1 subunit is a target of TGF-ß2 signaling, with a critical role in the RPE cell EMT process and suggests a possible mechanism through which its levels may be regulated during TGF-ß2 mediated EMT in ARPE-19 cells.